WO2010117010A1

WO2010117010A1 - Tumor marker and use thereof

Info

Publication number: WO2010117010A1
Application number: PCT/JP2010/056293
Authority: WO
Inventors: 慎吾一宮; 智樹菊地; 暁子外岡; 昇志佐藤
Original assignee: 北海道公立大学法人札幌医科大学
Priority date: 2009-04-10
Filing date: 2010-04-07
Publication date: 2010-10-14
Also published as: EP2418490A1; JPWO2010117010A1; CN102365551A; EP2418490A4; US20120129168A1

Abstract

SNX5 is detected in a sample collected from a subject. SNX5 can be used as a tumor marker specific to papillary thyroid carcinoma, whereby the diagnosis of papillary thyroid carcinoma can be achieved readily. A technique for detecting papillary thyroid carcinoma using the tumor marker is also provided.

Description

Tumor markers and their use

The present invention relates to a tumor marker and use thereof, and more particularly, to a tumor marker detection method suitable for diagnosis of papillary thyroid cancer and a tool used in the method.

Papillary thyroid cancer is the most frequently occurring malignant tumor among thyroid cancers, and it can have a poor prognosis if it metastasizes to the lungs or cervical lymph nodes. Therefore, a definitive diagnosis of papillary thyroid cancer is very important. Histopathological examination is indispensable for the definitive diagnosis of papillary thyroid cancer. However, because there are many malignant tumors with papillary structures other than papillary thyroid cancer, not only whether the lesion is primary, but also the tumorous lesions of lymph node metastasis are determined. It is very difficult.

The characteristics of the pathological tissue of papillary thyroid cancer are evaluated by the expression pattern of molecules specific to thyroid tissue. Known thyroid markers include thyroglobulin and thyroid transcription factor 1 (TTF-1). Thyroglobulin is a dimeric glycoprotein secreted from thyroid tissue and is known to increase in cells or blood in thyroid diseases.

WO2006 / 133361 (Internationally released on December 14, 2006) Special table 2008-500033 published (January 10, 2008)

The measurement of the amount of thyroglobulin in the blood is used to observe the progress of thyroid cancer after surgery (especially, the possibility of metastasis). However, from the measurement of thyroglobulin, it cannot be determined whether thyroid cancer is benign or malignant. Among the molecules that are reported to be specifically expressed in thyroid tissue, there are molecules that are also expressed in other organs. Therefore, an excellent tumor marker specific for papillary thyroid cancer is eagerly desired.

The present invention has been made in view of the above problems, and an object of the present invention is to provide a tumor marker specific for papillary thyroid cancer and a technique for discriminating thyroid papillary cancer using the tumor marker. .

As a result of producing an antibody against human SNX5 protein and performing immunohistochemical staining for various human normal tissues and neoplastic lesions using this anti-human SNX5 antibody, SNX5 was found in follicular epithelium of thyroid gland. Responds specifically and is particularly responsive to papillary thyroid cancer, a malignant tumor derived from the thyroid gland, as well as thyroid cancers of other histological types, papillary carcinoma derived from the digestive tract, and papillae derived from the lungs The present inventors have found that almost no expression of SNX5 is observed in thyroid carcinomas, and have completed the present invention.

That is, the method according to the present invention is a method for detecting a tumor marker, and includes a step of detecting SNX5 in a subject sample.

In the method according to the present invention, the subject sample is preferably a tissue collected from the subject or a culture or a slice thereof, but may be a cell lysate prepared from a tissue collected from the subject or a culture thereof. Good. The tissue is preferably a thyroid tissue, but may be a body fluid such as blood.

In the method according to the present invention, the subject is preferably a patient who may have thyroid disease, but may be a patient who has already developed papillary thyroid cancer.

The SNX5 is preferably a SNX5 protein or a fragment thereof. In this case, the step of detecting SNX5 is preferably an immunoassay using an anti-SNX5 antibody. The anti-SNX5 antibody used is preferably an anti-human SNX5 antibody, more preferably an anti-human SNX5 monoclonal antibody, and a mouse anti-human SNX5 monoclonal antibody produced from hybridoma 48C2 or a binding activity equivalent to the antibody. Most preferred is a monoclonal antibody having

The SNX5 may be a SNX5 gene or a fragment thereof. In this case, the step of detecting SNX5 is preferably a step of hybridizing a nucleic acid probe to the SNX5 gene or a fragment thereof. The nucleic acid probe is preferably a hybridization probe, but may be a PCR primer.

More preferably, the method according to the present invention includes both a step of detecting an SNX5 protein or a fragment thereof and a step of detecting an SNX5 gene or a fragment thereof.

The above method may be a method for providing a diagnostic criterion for papillary thyroid cancer, and may be a method for providing a criterion for differentiation of a malignant tumor exhibiting a papillary morphology. The above method may also be a method for providing a criterion for determining whether or not a lesion of papillary thyroid cancer is a primary lesion. Furthermore, the above method may be a method for providing a criterion for determining whether or not a tumor having metastasized to a cervical lymph node is derived from the thyroid gland.

The kit according to the present invention is a kit for detecting a tumor marker and is characterized by comprising an anti-SNX5 antibody. The kit according to the present invention preferably further comprises a reagent for detecting the antibody. The antibody is preferably an anti-human SNX5 antibody, more preferably an anti-human SNX5 monoclonal antibody, a mouse anti-human SNX5 monoclonal antibody produced from hybridoma 48C2, or a monoclonal antibody having a binding activity equivalent to the antibody. Most preferably.

The kit according to the present invention is a kit for detecting a tumor marker, and is characterized by comprising an oligonucleotide capable of hybridizing to the SNX5 gene or a fragment thereof. The kit according to the present invention preferably further comprises a reagent for detecting the oligonucleotide. The oligonucleotide is preferably a hybridization probe for the SNX5 gene or a fragment thereof, but may be a PCR primer.

The kit is preferably a kit for providing a diagnostic standard for papillary thyroid cancer, but may also be a kit for providing a criterion for identifying a malignant tumor exhibiting a papillary morphology. In addition, the kit may be a kit for providing a criterion for determining whether or not a lesion of papillary thyroid cancer is a primary lesion. Furthermore, the kit may be a kit for providing a criterion for determining whether or not a tumor with cervical lymph node metastasis originates from the thyroid gland.

Other objects, features, and superior points of the present invention will be fully understood from the following description. The advantages of the present invention will become apparent from the following description with reference to the accompanying drawings.

By using the present invention, it is possible to detect a tumor marker suitable for diagnosis of whether or not it is papillary thyroid cancer.

It is a figure which shows the result of the Western blot by an anti-human SNX5 monoclonal antibody with respect to the cell soluble fraction of the human 293 cell which introduce | transduced pCMV-SNX5. As a control, human 293 cells into which pCMV, pCMV-mutant AIRE # 1 or pCMV-mutant AIRE # 2 was introduced were used. It is a figure which shows the result of the immunohistochemical dyeing | staining by the anti- human SNX5 monoclonal antibody with respect to the human 293 cell which introduce | transduced pCMV-SNX5. As a control, human 293 cells into which pCMV had been introduced were used. All cells were fixed with paraformaldehyde. It is a figure which shows the result of the immunohistochemical dyeing | staining with the anti-human SNX5 monoclonal antibody with respect to the formalin fixed paraffin embedding section of a human adenocarcinoma tissue. (A) shows the results of immunohistochemical staining in papillary thyroid cancer, (b) shows the results of immunohistochemical staining in lung adenocarcinoma, and (c) shows the results of immunohistochemical staining in breast adenocarcinoma. . It is a figure which shows the result of the immunohistochemical dyeing | staining with an anti-human SNX5 polyclonal antibody with respect to the formalin fixed paraffin embedding section of a human adenocarcinoma tissue. It is a figure which shows the result of quantitative RT-PCR about the human SNX5 gene in the normal tissue and cancer tissue of a thyroid gland.

As described above, the present inventors have shown that SNX5 is highly expressed in papillary thyroid cancer, which is a malignant tumor derived from the thyroid gland, but thyroid cancer exhibiting other tissue types, papillary adenocarcinoma derived from the digestive tract It was found that almost no expression was observed in lung-derived papillary adenocarcinoma.

Human sorting nexin-5 (SNX5) is a molecule involved in intracellular endoplasmic reticulum transport and has been suggested to be closely related to cell migration (see, for example, Non-Patent Documents 1 and 2). Patent Document 1 lists many gene candidates related to multiple myeloma, and one of them is SNX5. Patent Document 2 lists many candidates for tumor-related peptides (antigen peptides) that bind MHC molecules, and one of them is a partial fragment of SNX5 protein (amino acids 292 to 300: SEQ ID NO: 524 of Patent Document 2). Is listed. However, the specificity of SNX5 in papillary thyroid cancer is not disclosed or suggested in any literature. Thus, the present invention provides an excellent tumor marker for papillary thyroid cancer.

[1. (Tumor marker)
The present invention provides an excellent tumor marker for papillary thyroid cancer. In one embodiment, the tumor marker of the present invention is SNX5 protein or a fragment thereof. In other embodiments, the tumor marker of the present invention is the SNX5 gene or a fragment thereof.

As used herein, a SNX5 protein is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; alternatively, one or several amino acids of the amino acid sequence shown in SEQ ID NO: 1 are deleted, substituted or A polypeptide comprising an added amino acid sequence and having SNX5 activity. The SNX5 protein is a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2; a nucleotide in which one or several bases of the nucleotide sequence shown in SEQ ID NO: 2 are deleted, substituted or added A polypeptide encoded by a polynucleotide comprising the sequence and having SNX5 activity; a polypeptide encoded by a polynucleotide capable of hybridizing under stringent conditions with the polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 2 and having SNX5 activity Or it may be a polypeptide encoded by a polynucleotide having a homology of 80% or more with the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2 and having SNX5 activity.

When used in terms of a polypeptide (amino acid), “one or several” is preferably in the range of 1 to 30, more preferably in the range of 1 to 20, and more preferably 1 to 10 More preferably, it is in the range of 1 to 5, and still more preferably in the range of 1 to 5. A person skilled in the art can easily understand the extent of the number of amino acids indicated by the term “one or several” depending on the length of the target polypeptide.

When used in terms of a polynucleotide (base), “one or several” is preferably in the range of 1 to 100, more preferably in the range of 1 to 50, and 1 to 30 More preferably, it is within the range of 1 to 15, and still more preferably within the range of 1 to 15. A person skilled in the art can easily understand the extent of the range of the number of bases indicated by the term “one or several” depending on the length of the target polynucleotide. For example, in the case of the oligonucleotide described below, the term “one or several” is preferably in the range of 1 to 10, more preferably in the range of 1 to 7, more preferably 1 to 5 More preferably, it is within the range, and still more preferably within the range of 1 to 3.

As used herein, the homology for the polypeptide or polynucleotide of interest is preferably 80% or more, more preferably 85% or more, and more preferably 90% or more. Preferably, it is more preferably 95% or more.

As used herein, the term “SNX5 activity” intends the ability to bind to an anti-SNX antibody elicited using a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 as an antigen. The SNX antibody may be an anti-SNX5 antibody described later, and a 48C2 antibody described later is preferable. The polypeptide may be a recombinant human SNX5 protein or an isolated and purified natural human SNX5 protein.

As used herein, the SNX5 gene is a polynucleotide that encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or alternatively one or several amino acids of the amino acid sequence shown in SEQ ID NO: 1 A polynucleotide encoding a polypeptide having an amino acid sequence deleted, substituted or added and having SNX5 activity. The SNX5 gene is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2, a nucleotide sequence in which one or several bases of the nucleotide sequence shown in SEQ ID NO: 2 are deleted, substituted or added, and SNX5 activity A polynucleotide encoding a polypeptide having SNX5 activity; a polynucleotide capable of hybridizing under stringent conditions with a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 2; Alternatively, it may be a polynucleotide that is a polynucleotide having a homology of 80% or more with the polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 2 and encoding a polypeptide having SNX5 activity.

SEQ ID NO: 1 is the amino acid sequence of SNX5 registered in GenBank (accession number AF121855), SEQ ID NO: 2 is the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1, and GenBank (accession number) AF121855) corresponds to the open reading frame (positions 181 to 1395) of the nucleotide sequence of SNX5 (SEQ ID NO: 3) registered in AF121855).

As used herein, the term “polypeptide” is used interchangeably with “peptide” or “protein” and is intended to be a polymer of amino acids. In addition, “fragment” of a polypeptide is intended to be a partial fragment of the polypeptide. The polypeptides according to the invention may also be produced recombinantly, chemically synthesized or isolated from natural sources.

As used herein, the term “polynucleotide” is used interchangeably with “gene”, “nucleic acid” or “nucleic acid molecule” and is intended to be a polymer of nucleotides. A “fragment” of a polynucleotide is intended to be a partial fragment of the polynucleotide. As used herein, the term “nucleotide sequence” is used interchangeably with “nucleic acid sequence” or “base sequence”.

The polynucleotide according to the present invention may exist in the form of RNA (for example, mRNA) or in the form of DNA (for example, cDNA or genomic DNA). DNA can be double-stranded or single-stranded. Single-stranded DNA or RNA can be the coding strand (also known as the sense strand) or it can be the non-coding strand (also known as the antisense strand).

As used herein, the term “oligonucleotide” is intended to be a combination of several to several tens of nucleotides, and is used interchangeably with “polynucleotide”.

As used herein, the term “stringent (hybridization) conditions” refers to a hybridization solution (50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM phosphorous. After overnight incubation at 42 ° C. in sodium acid (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA, Although it is intended to wash the filter in 1 × SSC, the washing conditions at high stringency are appropriately changed depending on the polynucleotide to be hybridized. For example, when using mammalian DNA, 0.1% SDS Washing at 65 ° C. in 0.5 × SSC containing (preferably 15 Min x 2) is preferred. When using E. coli-derived DNA, washing at 68 ° C. in 0.1 × SSC containing 0.1% SDS (preferably 15 minutes × 2 times) is preferable, and when using RNA, 0.1% Washing at 68 ° C. in 0.1 × SSC containing SDS (preferably twice for 15 minutes) is preferable, and when using oligonucleotide, in 0.1 × SSC containing 0.1% SDS Washing at the hybridization temperature (preferably 15 minutes × 2 times) is preferred.

By using the tumor marker of the present invention, it is possible to provide a criterion for determining a malignant tumor exhibiting papillary morphology, so that it is easy to diagnose whether or not it is papillary thyroid cancer. The present invention is very effective in determining whether or not a lesion is a primary lesion. Furthermore, by using the present invention, it is possible to more clearly determine whether a tumor that has metastasized to the cervical lymph node is derived from the thyroid gland or another tissue (lung, mammary gland, etc.).

[2. (Tumor marker detection method)
The present invention provides a method for detecting a tumor marker. The tumor marker detection method according to the present invention includes a step of detecting SNX5 in a subject sample. In one embodiment, the detection method according to the present invention includes a step of detecting SNX5 protein or a fragment thereof. In another embodiment, the detection method according to the present invention includes a step of detecting an SNX5 gene or a fragment thereof.

As used herein, a “subject sample” is intended any tissue (including bodily fluids such as blood) or cells collected from a subject, and tissue sections or cells prepared therefrom. Lysates can also be included in the subject sample. Preferred subject samples for use in the present invention include, but are not limited to, tumor tissue and serum. In addition, the process of taking out a tissue or a cell directly from a subject as a first stage of sample acquisition is performed by a doctor and is outside the scope of the present invention. In addition, the step of determining whether or not the disease is a disease using the result obtained by the method of the present invention is also performed by a doctor and is outside the scope of the present invention.

The subject to be the subject of the present invention is preferably a patient who may suffer from thyroid disease, more preferably a patient who develops papillary thyroid cancer, but may be a normal patient. In the method according to the present invention, the subject sample is thyroid tissue or a culture thereof collected from a patient who may have thyroid disease, or a tissue section or cell lysate prepared therefrom. However, it is not limited to this. Those skilled in the art will readily understand that the procedure for obtaining a sample can be appropriately selected depending on the desired tissue or cell.

In the embodiment of detecting SNX5 protein, the step of detecting SNX5 may be an immunoassay using an anti-SNX5 antibody. As used herein, the term “immunoassay” intends an assay performed utilizing an immunological binding reaction based on an antigen-antibody reaction. Assays that utilize immunological binding reactions include immunohistochemistry, immunoelectron microscopy, western blot, immunoprecipitation, sandwich ELISA assay, radioimmunoassay, and antibody assay (eg, immunodiffusion assay), and affinity chromatography Etc. These techniques are well known in the art, and those skilled in the art can easily carry out the present invention.

The anti-SNX5 antibody is preferably an anti-human SNX5 antibody, more preferably a monoclonal antibody, and more preferably a mouse anti-human SNX5 monoclonal antibody (also referred to as 48C2 antibody) produced from hybridoma 48C2. The present inventors have found that the 48C2 antibody recognizes the N-terminal side of human SNX5 protein (positions 1 to 177 of SEQ ID NO: 1: SEQ ID NO: 10) (results not shown). That is, the “SNX5 protein fragment” is a polypeptide consisting of a partial sequence of the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence consisting of 8 or more consecutive amino acids thereof. It is preferable that it is polypeptide containing. Those skilled in the art who have read this specification will easily understand that monoclonal antibodies having binding properties equivalent to those of the 48C2 antibody are also within the range of antibodies suitably used in the present invention.

When the subject sample is a tissue collected from the subject or a culture or section thereof, the immunoassay can be immunohistochemical staining. In the case of cell lysates prepared from tissue or cultures taken from a subject, the immunoassay may be a Western blot. Alternatively, if the subject sample is blood collected from the subject, the immunoassay can be an ELISA. These immunoassays are preferably used for differential diagnosis before surgery, but may be used for examining the presence or absence of metastasis after surgery.

The mouse anti-human SNX5 monoclonal antibody produced from the hybridoma 48C2 is not only suitable for detecting SNX5 protein by Western blotting or SNX5 protein in formalin-fixed paraffin sections, but also for identifying SNX5 protein in paraform-fixed tissues I made it. So far, the function of SNX5 in the cytoplasm has been reported. Even when this mouse anti-human SNX5 monoclonal antibody was used, it was shown that SNX5 was localized in the cytoplasm. This supports the specificity of this mouse anti-human SNX5 monoclonal antibody. Hybridoma 48C2 is maintained under the control of the Hokkaido University of Medicine Sapporo Medical University Intellectual Property Management Office (060-8556, Minami 1 Nishi 17-chome, Chuo-ku, Sapporo) and should be sold as needed. Is possible.

Immunohistochemical examination is very important when performing histopathological diagnosis of malignant tumors. By using a tumor marker specific to a specific organ or tissue, it becomes easy to make a definitive diagnosis of a metastatic lesion whose primary lesion has not yet been established, and a diagnosis or treatment policy can be quickly determined. Various organ-specific molecular markers have been found so far, but antibodies actually used for histopathological diagnosis are limited. In particular, an antibody suitable for differentiating papillary thyroid cancer has not yet been found.

When formalin-fixed paraffin sections used in normal histopathological examination were subjected to immunohistochemical staining using a mouse anti-human SNX5 monoclonal antibody, strong expression of SNX5 was observed in papillary carcinoma derived from the thyroid gland. No expression was observed in lung cancer or breast cancer-derived cancer tissues, which are typical examples of other adenocarcinomas. For the purpose of verifying this result, immunohistochemical staining using a commercially available rabbit anti-human SNX5 polyclonal antibody was performed. Similarly, high expression of SNX5 in tumor cells was observed. Thus, SNX5 is very excellent as a tumor marker for papillary thyroid cancer. The mouse anti-human SNX5 monoclonal antibody is a very excellent detection tool that enables immunohistochemical analysis of paraform-fixed tissues.

In the embodiment of detecting the SNX5 gene, the step of detecting SNX5 can be a step of hybridizing a nucleic acid probe to the SNX5 gene or a fragment thereof. As used herein, the “nucleic acid probe” is not particularly limited as long as it can hybridize to a target nucleic acid, and may be a so-called hybridization probe or a PCR primer. Hybridization techniques and PCR techniques are well known in the art and one of ordinary skill in the art can readily practice the invention. When the subject sample is a tissue sample collected from the subject, various techniques known in the art can be employed, for example, in situ hybridization technology, in situ PCR technology, or the like. When the subject sample is blood collected from the subject, for example, a conventional PCR technique can be employed.

The nucleic acid probe is not particularly limited as long as it is an oligonucleotide containing a partial sequence of the SNX5 gene (or its complementary strand), and may be an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 4 or 5 or its complementary sequence, It may be an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NOs: 6 to 9 or its complementary sequence. Further, since the 48C2 antibody recognizes the N-terminal side of the human SNX5 protein, the nucleic acid probe may be an oligonucleotide encoding the “SNX5 protein fragment”.

In the case of in situ hybridization, the nucleic acid probe is preferably an oligonucleotide consisting of 15 to 50 consecutive bases of the nucleotide sequence shown in SEQ ID NO: 1 or its complementary sequence, more preferably 20 to 50, and still more preferably An oligonucleotide consisting of 20 to 45, most preferably 25 to 40 consecutive bases. In the case of PCR (including in situ PCR), the nucleic acid probe is preferably an oligonucleotide consisting of 15 to 50 consecutive bases of the nucleotide sequence shown in SEQ ID NO: 1 or its complementary sequence, more preferably 15 An oligonucleotide consisting of ˜40, more preferably 15-35, most preferably 25-35 consecutive bases.

In addition, although the detection method according to the present embodiment includes the step of detecting the SNX5 gene, it is more preferable to further include the step of detecting the SNX5 protein described above.

As described above, by using the method for detecting a tumor marker according to the present invention, it is possible to provide a criterion for determining a malignant tumor exhibiting a papillary morphology, so that it is easy to diagnose whether or not it is papillary thyroid cancer. . The present invention is very effective in determining whether or not a lesion is a primary lesion. Furthermore, by using the present invention, it is possible to more clearly determine whether a tumor that has metastasized to the cervical lymph node is derived from the thyroid gland or another tissue (lung, mammary gland, etc.). That is, the method according to the present invention can also be a method that provides a diagnostic criterion for papillary thyroid cancer (for example, a method for obtaining data for diagnosing papillary thyroid cancer). It may also be a method of providing a criterion (for example, a method of acquiring data for distinguishing a malignant tumor exhibiting a papillary morphology). Further, the method according to the present invention provides a criterion for determining whether or not a lesion of papillary thyroid cancer is a primary lesion (for example, for determining whether or not a lesion of papillary thyroid cancer is a primary lesion). It can also be a method of acquiring data. Furthermore, the method according to the present invention provides a criterion for determining whether or not a tumor with cervical lymph node metastasis originates from the thyroid gland (for example, whether or not a tumor with cervical lymph node metastasis originates from the thyroid gland). It may also be a method of acquiring data for determination.

[3. (Tumor marker detection tool)
The present invention provides a kit for detecting a tumor marker. The kit according to the present invention is characterized by comprising a tool for detecting SNX5 in a subject sample.

As used herein, the term “kit” is intended as a package with a container (eg, bottle, plate, tube, dish, etc.) containing a particular material, but as a composition. Forms containing the material in the substance are also encompassed by the term “kit”. The kit preferably includes instructions for using each material. As used herein, in the aspect of a kit, “comprising” is intended to mean being contained in any of the individual containers that make up the kit. Moreover, the kit which concerns on this invention may be the packaging which packed several different compositions in one, and in the case of a solution form, you may enclose in the container. The kit according to the present invention may comprise substance A and substance B mixed in the same container or in separate containers. The “instructions” may be written or printed on paper or other media, or may be affixed to electronic media such as magnetic tape, computer readable disk or tape, CD-ROM, etc. . The kit according to the present invention may also include a container containing a diluent, a solvent, a washing solution or other reagent. Furthermore, the kit according to the present invention may include instruments and reagents necessary for collecting a subject sample. In addition, the kit according to the present invention may be equipped with instruments and reagents necessary for preparing a section or cell lysate from a subject sample.

In one embodiment, the kit according to the present invention includes an antibody for detecting SNX5 protein or a fragment thereof. The antibody is preferably an anti-human SNX5 antibody, more preferably an anti-human SNX5 monoclonal antibody, a mouse anti-human SNX5 monoclonal antibody produced from hybridoma 48C2, or a monoclonal antibody having a binding activity equivalent to the antibody. Most preferably. The kit according to the present invention preferably further comprises a reagent for detecting the above-described antibody, but as described above, a composition containing an antibody for detecting the SNX5 protein or a fragment thereof. May be provided as

In another embodiment, the detection kit according to the present invention includes an oligonucleotide for detecting the SNX5 gene or a fragment thereof. The oligonucleotide is preferably an oligonucleotide that can hybridize to the SNX5 gene or a fragment thereof, and is preferably a hybridization probe or a PCR primer for the SNX5 gene or a fragment thereof. The kit according to the present invention preferably further comprises a reagent for detecting the oligonucleotide, but as described above, it contains an oligonucleotide for detecting the SNX5 gene or a fragment thereof. It may be provided as a composition.

The kit according to the present invention is preferably a kit for providing a diagnostic criterion for papillary thyroid cancer, but may also be a kit for providing a criterion for identifying a malignant tumor exhibiting a papillary morphology. The kit according to the present invention may also be a kit for providing a criterion for determining whether or not a lesion of papillary thyroid cancer is a primary lesion. Furthermore, the kit according to the present invention may be a kit for providing a criterion for determining whether or not a tumor that has metastasized to a cervical lymph node is derived from the thyroid gland.

Thus, an object of the present invention is to provide a tumor marker and to provide a diagnostic criterion for papillary thyroid cancer by detecting the tumor marker. Those skilled in the art who have read this specification will readily understand that the present invention can be implemented in various forms based on the description and common general knowledge in this specification.

[1. Acquisition of human SNX5 cDNA]
Using total RNA extracted from human HacaT cells as a template, forward primer (pCMV-HA-SNX5 FW: 5'-CAGGCCCGAATTCGGATGGCCGCGGTTCCCGAG-3 '(SEQ ID NO: 4)) and reverse primer (pCMV-HA-SNX5 RV: 5'- RT-PCR using GATCTCGGTCGACCGTGAAGGCATATCAGTTAT-3 ′ (SEQ ID NO: 5)) was performed to obtain human SNX5 cDNA. The obtained human SNX5 cDNA was inserted into an expression vector for Escherichia coli pET3c (Novagen) and a mammalian expression vector pCMV-HA (BD Bioscience) to prepare pET3c-SNX5 and pCMV-HA-SNX5, respectively. Note that pCMV-HA-SNX5 FW is obtained by ligating a restriction enzyme site or the like to the sequence of the 5 ′ region of human SNX5 cDNA (ATGGCCGCGGTTCCCGAG (SEQ ID NO: 6)), and pCMV-HA-SNX5 RV is human SNX5 cDNA. A restriction enzyme site or the like is ligated to the complementary sequence of the 3 ′ region sequence (ataactgatatgccttcac (SEQ ID NO: 7)).

[2. Preparation of anti-human SNX5 monoclonal antibody]
E. coli BL21 introduced with pET3c-SNX5 was cultured, and protein synthesis was induced by IPTG. Subsequently, human SNX5 protein was separated / concentrated from the bacterial cell components. The obtained protein was used as an immunogen, and 6-8 week old Balb / c mice were immunized intraperitoneally (100 μg) using complete adjuvant (primary immunization only) or incomplete adjuvant (second and subsequent immunizations) as an adjuvant. /individual). Booster immunization was performed every other week for 2 months, and final immunization was performed 3 days before the collection of splenocytes. The collected spleen cells were fused with NS0 mouse myeloma cells using polyethylene glycol, and the fused cells were cultured in a 96-well plate in RPMI1640 medium containing HAT and 10% FBS for 2 to 3 weeks. The anti-human SNX5 monoclonal antibody was screened by Western blotting using the culture supernatant.

[3. Gene transfer to mammalian cells)
PCMV-SNX5 was introduced into human 293 cells cultured in DMEM containing 10% FBS and penicillin / streptomycin according to the manufacturer's instructions. In addition, the expression of SNX5 in the transformant was performed using the total RNA extracted from the transformant as a template, the forward primer (SNX5 AMP FW: 5'-ccggttaaagagcaaagacg-3 '(SEQ ID NO: 8)) and the reverse primer (SNX5 AMP It was confirmed by RT-PCR using RV: 5′-agctctgcaaaagggagaca-3 ′ (SEQ ID NO: 9)).

[4. Western blot analysis
Human 293 cells into which pCMV-SNX5 had been introduced were cultured for 3 days and then solubilized using a lysis buffer containing 0.5% NP-40. Proteins contained in the solubilized fraction were separated by SDS-PAGE using a 5-20% gradient gel. The separated protein was transferred to a nylon membrane, and a primary antibody reaction with an anti-human SNX5 monoclonal antibody and a secondary antibody reaction with a peroxidase-conjugated goat anti-mouse IgG antibody were each performed for 1 hour. Signals were visualized using an ECL kit (Amersham).

As shown in FIG. 1, although a non-specific band was detected at a position of 50 kDa, a signal specific to SNX5 (molecular weight 47 kDa) was detected only in cells into which pCMV-SNX5 was introduced. Similar signals were not observed in cells transfected with negative controls (CMV, pCMV-mutant AIRE # 1, pCMV-mutant AIRE # 2).

[5. Immunohistochemical staining)
Human 293 cells introduced with pCMV-SNX5 were cultured for 3 days and then fixed with paraformaldehyde. Anti-human SNX5 monoclonal antibody was used as the primary antibody, and goat anti-mouse IgG antibody complexed with Alexa596 was used as the secondary antibody. And allowed to react at room temperature for 1 hour, and immunohistochemical staining was performed. Signals were detected using an IX71 fluorescence microscope (Olympus). FIG. 2 shows a cell into which pCMV-SNX5 was introduced (left) and a cell into which pCMV was introduced as a negative control (right). It can be seen that the anti-human SNX5 monoclonal antibody specifically reacts with SNX5.

In addition, immunohistochemical staining with an anti-human SNX5 monoclonal antibody was performed using formalin-fixed paraffin-embedded sections of adenocarcinoma tissue. Signals were detected using an automatic immunostaining device (manufactured by DAKO) (FIG. 3). As shown in FIG. 3 (a), strong expression of SNX5 was observed in papillary thyroid cancer. However, no expression of SNX5 was observed in lung adenocarcinoma (FIG. 3 (b)) and breast adenocarcinoma (FIG. 3 (c)) (FIG. 3 (b)).

Furthermore, the SNX5 expression distribution in the tumor tissue examined using the established anti-human SNX5 monoclonal antibody was verified using a commercially available rabbit anti-human SNX5 polyclonal antibody (H40: Santa Cruz Biotechnology) (FIG. 4). As shown in FIG. 4, in immunohistochemical staining using formalin-fixed paraffin sections of papillary thyroid cancer, strong expression of SNX5 was observed in tumor cells.

Furthermore, the expression of SNX5 in various malignant tumors showing papillary proliferation was analyzed using a mouse anti-human SNX5 monoclonal antibody. The results are shown in Table 1. As shown in Table 1, in all 20 cases of epithelial malignant tumors (including gastric cancer, colorectal cancer and pancreatic cancer) other than the thyroid gland, and 5 cases of sarcoma / non-epithelial malignant tumors, expression of SNX5 is completely observed. There wasn't.

[6. Quantitative PCR
Total RNA was extracted from normal thyroid tissue obtained by biopsy from a subject or thyroid cancer tissue removed by surgery from a patient who developed thyroid cancer, and cDNA was prepared using reverse transcriptase (Invitrogen) . Subsequently, quantitative PCR (Applied Biosystems) using a PCR probe (Product No .: Hs00429583) was performed using this cDNA as a template. The obtained values were analyzed using ribosomal RNA as a control and compared by the delta delta CT method (ABI 7000, Applied Biosystems). In addition, the gene analysis of human tissue was limited to the expression analysis of SNX5, and the use of human tissue was conducted through an ethics committee under sufficient informed consent. We took great care to protect the data and protected human rights and legal compliance.

The results for 3 cases of thyroid cancer are shown in FIG. As shown in the figure, it was confirmed that the expression of the SNX5 gene was higher in the thyroid cancer tissue than in the normal thyroid tissue.

Thus, the present inventors found for the first time that SNX5 is highly expressed in papillary thyroid cancer. In addition, the mouse anti-human SNX5 monoclonal antibody established by the present inventors has enabled immunohistochemical analysis of not only formalin-fixed paraffin sections but also paraform-fixed tissues. The knowledge obtained by the present invention is that SNX5 is considered to be an excellent marker compared with known thyroid markers such as thyroglobulin and TTF-1, and can provide useful information in pathological diagnosis. Conceivable. In addition, serodiagnosis of neoplastic lesions derived from the thyroid gland is considered possible by ELISA using an anti-human SNX5 monoclonal antibody.

The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

In addition, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

The use of the present invention facilitates early diagnosis and definitive diagnosis of papillary thyroid cancer. The present invention providing such an excellent tool can be used in the fields of medicine and pharmacy and can greatly contribute to the development of pharmaceuticals and biochemical reagents.

Claims

A method for detecting a tumor marker for papillary thyroid cancer, comprising a step of detecting SNX5 protein or a fragment thereof in a subject sample.
The method according to claim 1, wherein the step is performed by an immunoassay using an anti-SNX5 antibody.
3. The method according to claim 2, wherein the anti-SNX5 antibody is a mouse anti-human SNX5 monoclonal antibody produced from hybridoma 48C2 or a monoclonal antibody having a binding activity equivalent to that of the antibody.
A method for detecting a tumor marker for papillary thyroid cancer, comprising a step of detecting an SNX5 gene or a fragment thereof in a subject sample.
The method according to claim 4, wherein the step is performed by hybridizing a nucleic acid probe to the SNX5 gene or a fragment thereof.
6. The method according to claim 5, wherein the nucleic acid probe is an oligonucleotide having a nucleotide sequence represented by any of SEQ ID NOs: 4 to 9 or a complementary sequence thereof.
The method according to any one of claims 1 to 6, wherein the subject sample is a tissue or a cell lysate prepared from a tissue collected from the subject or its cells.
The method according to claim 7, wherein the tissue is thyroid tissue.
The method according to any one of claims 1 to 8, wherein the subject is a patient who may have a thyroid disease.
A kit for detecting a tumor marker of papillary thyroid cancer, comprising an anti-SNX5 antibody.
The kit according to claim 10, wherein the anti-SNX5 antibody is a mouse anti-human SNX5 monoclonal antibody produced from hybridoma 48C2 or a monoclonal antibody having a binding activity equivalent to the antibody.
A kit for detecting a tumor marker of papillary thyroid cancer, comprising an oligonucleotide capable of hybridizing to the SNX5 gene or a fragment thereof.
The kit according to claim 12, wherein the oligonucleotide comprises the nucleotide sequence shown in any one of SEQ ID NOs: 4 to 9 or a complementary sequence thereof.